Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells

Photorhabdus luminescens Tc toxins consist of the cell‐binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP‐ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP‐ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP‐ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP‐ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.     

TcdA1 of Photorhabdus luminescens: Electrophysiological Analysis of Pore Formation and Effector Binding

Tc toxins are widely distributed among different gram-negative and gram-positive bacteria, where they act as pathogenicity factors. The toxins are composed of different components that form oligomers for biological activity. Lipid bilayer experiments were performed with the TcdA1 component of the Tc toxin from Photorhabdus luminescens, which preferentially kills insects by actin polymerization. TcdA1 was able to increase the specific conductance of artificial lipid bilayer membranes by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 125 pS in 150 mM KCl and were found to be cation selective. The single-channel conductance of the TcdA1-channels was only moderately dependent on the bulk aqueous KCl concentration, which indicated point-charge effects on the channel properties. Experiments to study the voltage dependence of the TcdA1 channel demonstrated that it is reconstituted in a fully oriented way when it is added to only one side of the lipid bilayer membrane. A combination of biologically active components (TccC3) and a possible chaperone (TcdB2) blocked the TcdA1-mediated conductance efficiently in a dose-dependent manner when they were added to the cis side of the membrane. The half-saturation constant for binding of TcdB2-TccC3 to TcdA1 is in the low nanomolar range.     

The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A

The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C-terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA(1-1874)) and found that this mutant was still cytopathic. However, TcdA(1-1874) possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP-mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA(1-1874) to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA(1-1874) exhibits considerably higher cytotoxic potency than apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.     

Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A

Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.     

Putative toxins from the entomopathogenic bacterium Photorhabdus luminescens kill Armadillidium vulgare (Terrestrial isopod)

Terrestrial isopods can be killed by some entomopathogenic bacteria among Xenorhabdus and Photorhabdus species even with no or very limited multiplication. This suggests that toxemia and not septicemia is the major cause of entomopathogenic bacteria pathogenicity against these crustaceans. In this paper, we revealed that the injection of stationary phase culture supernatant of P. luminescens TT01, in which toxins can be accumulated, led alone to a rapid decrease in the number of host immune cells and killed most of the Armadillidium vulgare individuals within 48 h. The pathogenicity was strongly attenuated when supernatant was heated and totally suppressed after 100-kDa filtration suggesting that the toxin responsible for killing A. vulgare would be a protein above this size. Additionally, we tested the culture supernatant of two TT01 mutants that have been previously shown as being altered in their pathogenicity against lepidopteran insects one of them being known as exhibiting lower expression of some toxins. However, the supernatants of the mutants was as pathogenic for A. vulgare as the wild type strains suggesting that the toxins involved in killing A. vulgare may be different than previously described ones.     

The chaperone Hsp90 and PPIases of the cyclophilin and FKBP families facilitate membrane translocation of Photorhabdus luminescens ADP‐ribosyltransferases

TccC3 and TccC5 from Photorhabdus luminescens are ADP‐ribosyltransferases, which modify actin and Rho GTPases, respectively, thereby inducing polymerization and clustering of actin. The bacterial proteins are components of the Photorhabdus toxin complexes, consisting of the binding and translocation component TcdA1, a proposed linker component TcdB2 and the enzymatic component TccC3/5. While the action of the toxins on target proteins is clearly defined, uptake and translocation of the toxins into the cytosol of target cells are not well understood. Here we show by using pharmacological inhibitors that heat shock protein 90 (Hsp90) and peptidyl prolyl cis/trans isomerases (PPIases) including cyclophilins and FK506‐binding proteins (FKBPs) facilitate the uptake of the ADP‐ribosylating toxins into the host cell cytosol. Inhibition of Hsp90 and/or PPIases resulted in decreased intoxication of target cells by Photorhabdus toxin complexes determined by cell rounding and reduction of transepithelial electrical resistance of cell monolayers. ADP‐ribosyltransferase activity of toxins and toxin‐induced pore formation were notimpaired by the inhibitors of Hsp90 and PPIases. The Photorhabdus toxins interacted with Hsp90, FKBP51, Cyp40 and CypA, suggesting a role of these host cell factors in translocation and/or refolding of the ADP‐ribosyltransferases.     

TcA, the putative transposase of the C. elegans Tc1 transposon, has an N-terminal DNA binding domain.

Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion protein of beta-galactosidase and TcA also exhibits DNA binding; deletion derivatives of this fusion protein were tested for DNA binding. A deletion of 39 amino acids at the N-terminal region of TcA abolishes the DNA binding, whereas a deletion of 108 C-terminal amino acids does not affect DNA binding. This shows that the DNA binding domain of TcA is near the N-terminal region. The DNA binding capacity of TcA supports the assumption that TcA is a transposase of Tc1.     

Clostridium difficile toxins A and B: Receptors,pores, and translocation into cells

The most potent toxins secreted by pathogenic bacteria contain enzymatic moieties that must reach the cytosol of target cells to exert their full toxicity. Toxins such as anthrax, diphtheria, and botulinum toxin all use three well-defined functional domains to intoxicate cells: a receptor-binding moiety that triggers endocytosis into acidified vesicles by binding to a specific host-cell receptor, a translocation domain that forms pores across the endosomal membrane in response to acidic pH, and an enzyme that translocates through these pores to catalytically inactivate an essential host cytosolic substrate. The homologous toxins A (TcdA) and Toxin B (TcdB) secreted by Clostridium difficile are large enzyme-containing toxins that for many years have eluded characterization. The cell-surface receptors for these toxins, the non-classical nature of the pores that they form in membranes, and mechanism of translocation have remained undefined, exacerbated, in part, by the lack of any structural information for the central ～1000 amino acid translocation domain. Recent advances in the identification of receptors for TcdB, high-resolution structural information for the translocation domain, and a model for the pore have begun to shed light on the mode-of-action of these toxins. Here, we will review TcdA/TcdB uptake and entry into mammalian cells, with focus on receptor binding, endocytosis, pore formation, and translocation. We will highlight how these toxins diverge from classical models of translocating toxins, and offer our perspective on key unanswered questions for TcdA/TcdB binding and entry into mammalian cells.     

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.     

Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.     

The Interaction of N-Glycans in Fcγ Receptor I α-Chain with Escherichia coli K1 Outer Membrane Protein A for Entry into Macrophages: EXPERIMENTAL AND COMPUTATIONAL ANALYSIS*

Neonatal meningitis, caused by Escherichia coli K1, is a serious central nervous system disease. We have established that macrophages serve as permissive niches for E. coli K1 to multiply in the host and for attaining a threshold level of bacterial load, which is a prerequisite for the onset of the disease. Here, we demonstrate experimentally that three N-glycans in FcγRIa interact with OmpA of E. coli K1 for binding to and entering the macrophages. Adoptive transfer of FcγRIa^−/− bone marrow-derived macrophages transfected with FcγRIa into FcγRIa^−/− newborn mice renders them susceptible to E. coli K1-induced meningitis. In contrast, mice that received bone marrow-derived macrophages transfected with FcγRIa in which N-glycosylation sites 1, 4, and 5 are mutated to alanines exhibit resistance to E. coli K1 infection. Our molecular dynamics and simulation studies predict that N-glycan 5 exhibits strong binding at the barrel site of OmpA formed by loops 3 and 4, whereas N-glycans 1 and 4 interact with loops 1, 3, and 4 of OmpA at tip regions. Molecular modeling data also suggest no role for the IgG binding site in the invasion process. In agreement, experimental mutations in IgG binding site had no effect on the E. coli K1 entry into macrophages in vitro or on the onset of meningitis in newborn mice. Together, this integration of experimental and computational studies reveals how the N-glycans in FcγRIa interact with the OmpA of E. coli K1 for inducing the disease pathogenesis.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria

The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.     

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.     

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le^x-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le^x as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.     

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.     

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 Å x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a β-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a π-cation interaction within the β-flap that appear to be unique among the large clostridial cytotoxins.Clostridium difficile is a Gram-positive, spore-forming anaerobe that infects the colon and causes a range of disorders, including diarrhea, pseudomembranous colitis, and toxic megacolon (1, 2). Two large toxins, TcdA² and TcdB (308 and 270 kDa, respectively) are recognized as the main virulence factors of C. difficile, although their relative importance is the subject of on-going study (3, 4). These proteins belong to a class of homologous toxins called large clostridial toxins (LCTs) and have been classified more broadly as AB toxins, wherein a B moiety is involved in the delivery of an enzymatic A moiety into the cytosol of a target cell. In LCTs, the A subunit is an N-terminal glucosyltransferase that inactivates small G-proteins, such as Rho, leading to cell rounding and apoptosis of the intoxicated cell (5, 6). The B subunit corresponds to the remainder of the toxin and is responsible for binding the target cell through a C-terminal receptor-binding domain (7–9) and forming the membrane pore needed for translocation of the A subunit (10, 11). Unlike other known AB toxins, the glucosyltransferase A domains of LCTs are released from the B subunits by an autoproteolytic cleavage event (12). Cleavage is triggered by host inositol phosphates and the reducing environment of the cytosol (12).In LCTs, autoproteolysis has been attributed to a cysteine protease activity located within the N-terminal region of the B subunit (13). This region was identified based on homology with the cysteine protease domain (CPD) found in the multifunctional autoprocessing repeats in toxins (MARTX) toxins from Gram-negative bacteria (14). Autoprocessing in the MARTX toxin from Vibrio cholera (VcRTx) is also stimulated by InsP6 (15). A recent crystal structure of VcRTx CPD bound to InsP6 suggests a novel mechanism of InsP6-induced allosteric activation (16). The CPDs of TcdA and VcRTx share only 19% sequence identity. To gain insight into the mechanistic commonalities between these entirely different toxins and to delineate the LCT-specific modes of InsP6-induced processing, we performed structural and functional analyses on the cysteine protease from TcdA.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.